Ca2+ regulation of contraction will be investigated in muscle from the barnacle, Balanus nubilus, from which we have learned much over the past 30 years. In this application to renew a long-lasting, productive grant, the investigators propose to investigate in barnacle muscle fibers 1) how length and cross-bridge attachment modify Ca2+ binding to TnC in these long sarcomere length muscles, 2) whether cross-bridge attachment modifies the off rate of Ca2+ dissociation from TnC measured in fibers, 3) whether this provides a mechanism to couple Ca2+ dissociation and cross-bridge dissociation during relaxation, 4) whether barnacle muscle is dual regulated, 5) changes in TnC structure on activation, and 6) the role of Ca2+ binding to TnC in stabilizing TnC binding to the thin filament and in filament activation. They will use measurements of force in skinned fibers to assess Ca2+ activation, fluorescence of native or recombinant TnC in isolation or exchanged into skinned fibers to measure Ca2+ binding, stopped-flow measurements and activation of caged chelators to assess Ca2+ binding kinetics in isolated TnC or in TnC exchanged into fibers, site-directed mutagenesis of barnacle TnC to test molecular models of regulation, laser confocal microscopy to localize Ca2+ binding in the sarcomere, in vitro motility techniques and extraction of myosin regulatory light chains to test dual regulation. With these results on thin and thick filament regulation and effects of length and cross-bridges on the Ca2+ binding and activation, we will construct a more complete model of activation of contraction. This is important to aid the understanding of Ca2+ regulation of contraction of muscle in general as the mechanisms in other muscles are similar to those in barnacles. In barnacles, the size of individual fibers and sarcomeres allows more parameters to be measured and hypotheses tested. This will provide a more complete understanding of how muscle contraction is regulated, the central step in movement, and how movement itself affects regulation.